Hi!. I'm figuring out how to induce cellular stress by DNA damage. I'm working with HeLa and HEK 293T cells, and I need to study the effects of the CHK2 kinase pathway that phosphorylates a protein (hnRNP A1). I know that I can use UV radiation for this, and I wonder if the UV light of the culture hood could work. I know that it could be difficult to control because it depends on the magnitude of the radiation, but do you know if it can be set to make it work? or if there is some easier method?
Thank you!
Hi,
if the mode of action does not matter, we made good experience with DNA-alkylating agents (ethyl and methyl methanesulfonate). Radical induced oxidative stress can be induced via hydrogen peroxide, or preferrably with a radical inducer such as menadione or rotenone (more consistent damage). Mitomycin C is another freqently used positive control for DNA damage. Finally, UV can be an excellent choice, albeit hard to reproduce and/or accurately dose without any means to quantify dose, as you noted yourself. If you have an UV crosslinker in your lab, those are much better for accurate UV irradiation. Some elegant approaches for when the modes of action of the already mentioned approches may interfere with particular research questions are based on transfer of restriction enzymes to the nucleus to get direct DNA fragmentation.
Kind regards
- Badges
- Science topic
- Similar topics
- Chemistry
- Pharmacology
- Pharmaceuticals
- Drugs